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Structure-guided AAV capsid evolution strategies for enhanced CNS gene delivery

Nature Protocols volume 18, pages 3413–3459 (2023)Cite this article

Subjects

Abstract

Over the past 5 years, our laboratory has systematically developed a structure-guided library approach to evolve new adeno-associated virus (AAV) capsids with altered tissue tropism, higher transduction efficiency and the ability to evade pre-existing humoral immunity. Here, we provide a detailed protocol describing two distinct evolution strategies using structurally divergent AAV serotypes as templates, exemplified by improving CNS gene transfer efficiency in vivo. We outline four major components of our strategy: (i) structure-guided design of AAV capsid libraries, (ii) AAV library production, (iii) library cycling in single versus multiple animal models, followed by (iv) evaluation of lead AAV vector candidates in vivo. The protocol spans ~95 d, excluding gene expression analysis in vivo, and can vary depending on user experience, resources and experimental design. A distinguishing attribute of the current protocol is the focus on providing biomedical researchers with 3D structural information to guide evolution of precise ‘hotspots’ on AAV capsids. Furthermore, the protocol outlines two distinct methods for AAV library evolution consisting of adenovirus-enabled infectious cycling in a single species and noninfectious cycling in a cross-species manner. Notably, our workflow can be seamlessly merged with other RNA transcript-based library strategies and tailored for tissue-specific capsid selection. Overall, the procedures outlined herein can be adapted to expand the AAV vector toolkit for genetic manipulation of animal models and development of human gene therapies.

Key points

  • This protocol uses structure-guided design of AAV capsid libraries, infectious and noninfectious library cycling and evaluation of lead candidates in vivo to evolve new AAV vectors for enhanced CNS gene delivery.

  • This approach evolves novel AAV variants iteratively across multiple species. The vectors produced can display altered tissue tropism, higher transduction efficiency and the ability to evade pre-existing humoral immunity.

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Fig. 1: Methods for AAV evolution and in vivo characterization.
Fig. 2: Structural modeling of wild-type AAV serotypes used as the backbone for directed evolution.
Fig. 3: Generation of the AAV capsid library plasmid.
Fig. 4: NGS library preparation.
Fig. 5: GFP reporter gene expression in C57/BL6 mouse brain after ICV injection.
Fig. 6: Transduction of 4.69 and AAV4 packaging CMV-Cre after ICV dosing of mice.
Fig. 7: GFP reporter gene expression in C57/B6 mouse brain after i.v. administration of ccAAV vectors.
Fig. 8: IntraCSF dosing of ccAAV vectors in the pig CNS.
Fig. 9: IntraCSF dosing of AAV.cc84 or AAV9 after ICV injection in mice.
Fig. 10: Structural modeling of evolved variant AAVs.

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Data availability

The example data generated for the protocol has not been previously published; however, the protocol has previously been used to produce similar results shown in refs. 12,13,14. All data associated with the figures of this study are included in this paper. Any additional data to support these findings can be made available upon reasonable request to the corresponding author. Source data are provided with this paper.

Code availability

The Perl scripts used for NGS analysis of AAV variants have been described previously12. The scripts have been deposited into the Zenodo repository and can be accessed via https://doi.org/10.5281/zenodo.7075694.

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Acknowledgements

We acknowledge L. Edwards and K. Gleason for their assistance in completing in-life portions for our pig studies, N. Olby for her assistance in performing the IT infusions in pigs, J. McNamara and E. Matthews for their assistance with dissection of pig brains and Duke DLAR for their assistance with mouse care. We acknowledge the Duke light microscopy core facility and Translating Duke Health initiative for their support. Figures were created in part by using BioRender.

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Author notes

  1. These authors contributed equally: Trevor J. Gonzalez, Aaron Mitchell-Dick.

Authors and Affiliations

  1. Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC, USA

    Trevor J. Gonzalez & Aravind Asokan

  2. Department of Surgery, Duke University School of Medicine, Durham, NC, USA

    Aaron Mitchell-Dick, Leo O. Blondel, Marco M. Fanous, Joshua A. Hull, Daniel K. Oh & Aravind Asokan

  3. Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Sven Moller-Tank & Ruth M. Castellanos Rivera

  4. North Carolina State University College of Veterinary Medicine, Raleigh, NC, USA

    Jorge A. Piedrahita

  5. Department of Biomedical Engineering, Duke University, Durham, NC, USA

    Aravind Asokan

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Contributions

T.J.G., A.M.-D. and A.A. conceptualized and wrote the manuscript. T.J.G. and J.A.H. conducted the structural modeling. T.J.G. and S.M.-T. designed and cloned the AAV libraries. T.J.G., A.M.-D., S.M.-T. and R.M.C.R. optimized viral production and purification protocols and conducted in vivo cycling. T.J.G., A.M.-D. and A.A. designed experiments and created the figures. T.J.G., A.M.-D., L.O.B., M.M.F. and D.K.O. conducted experiments and analyzed data. A.A. and J.A.P. acquired funding.

Corresponding author

Correspondence to Aravind Asokan.

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Competing interests

T.J.G. and A.A. have filed patent applications on the subject matter of this paper. A.A. is a co-founder at StrideBio and TorqueBio and serves on the advisory boards of Atsena Therapeutics, Isolere Bio, Mammoth Bio, Ring Therapeutics and Kriya Therapeutics. S.M.-T. is a current employee of Regeneron Pharmaceuticals. R.M.C.R. is a current employee of Kriya Therapeutics.

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Nature Protocols thanks David Schaffer and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Tse, L. V. et al. Proc. Natl. Acad. Sci. USA 114, E4812–E4821 (2017): https://doi.org/10.1073/pnas.1704766114

Havlik, L. P. et al. J. Virol. 94, e00976-20 (2020): https://doi.org/10.1128/JVI.00976-20

Gonzalez, T. J. et al. Nat. Commun. 13, 5947 (2022): https://doi.org/10.1038/s41467-022-33745-4

Extended data

Extended Data Fig. 1 Representative Sanger sequencing traces for library quality control.

a, Representative sequencing traces of wild-type AAV9 VR VIII. Samples that match this would be excluded from library insert generation. b, Representative sequencing traces for AAV9 VR VIII containing high diversity at the library site. Users should confirm that the wild-type sequence is not called as the majority population and confirm overlapping histogram peak base calls at each position as shown. Samples that match this would be included during library insert generation. Figure created with BioRender.com.

Extended Data Fig. 2 Generation of iodixanol gradients for AAV purification.

Gradients can be created either by overlaying (a) or underlaying (b) of different densities in 17-ml ultracentrifuge tubes. These densities include 17%, 25%, 40% and 60% iodixanol. Virus is added on top of the completed gradient before spinning in an ultracentrifuge. Either method can be used successfully, and the choice of method is based on user preference. Figure created with BioRender.com.

Extended Data Fig. 3 ICV injection station.

a, Ice anesthetization setup. Two large gloves are filled with ice to create a sandwich chamber. When ready to begin, pups are placed between the two gloves of ice and incubated for 3–5 min. During this time, the pup will become unresponsive, and a loss of its pink body color will be observed. b, Stereotaxic injection apparatus with injection pump and Hamilton syringe. Once pups are anesthetized, mount pups on the stage and zero out your X and Y coordinates by moving your needle above the lambda point. The injection site is located 0.8 mm (X), followed by 1.5 mm (Y) up toward the coronal suture and −1.5 mm (Z).

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Source Data Fig. 5

Statistical source data for panels b, c and d

Source Data Fig. 6

Statistical source data for panel h

Source Data Fig. 7

Statistical source data for panels b and c

Source Data Fig. 9

Statistical source data for panel b

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Gonzalez, T.J., Mitchell-Dick, A., Blondel, L.O. et al. Structure-guided AAV capsid evolution strategies for enhanced CNS gene delivery. Nat Protoc 18, 3413–3459 (2023). https://doi.org/10.1038/s41596-023-00875-y

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